In previous studies we identified a pedigreed breeding colony of feral Mus musculus musculus (designated Czech II) which does not contain mouse mammary tumor virus (MMTV) proviral genomes in their germline. We have now completed a study of the affect of the chemical carcinogen dimethyl-benzanthracene (DMBA) on the incidence of mammary gland neoplasia in Czech II mice. Three percent of the breeding females have developed mammary tumors whereas no tumors have been observed in virgin females up to two years of age. Treatment of the mice with DMBA significantly increased the frequency of tumors (30-50 percent of the treated mice) and decreased the latency in tumor development (average 11 months). Most of the chemically induced and all of the spontaneous mammary tumors were type A adenocarcinomas. In an independent study we obtained evidence that some lactating females contain MMTV gp52 envelop protein in their milk. Analysis of tumor cellular DNA revealed the presence of MMTV proviral DNA in many but not all mammary tumors. The corresponding liver cellular DNA from MMTV positive tumor bearing mice lacked MMTV proviral DNA. This suggests to us that the Czech II colony is infected with an exogenous MMTV. Restriction enzyme analysis of the MMTV proviral genomes in the mammary tumor cellular DNA showed that the viral genome was not that of common laboratory strains of MMTV since it contains several restriction site polymorphisms. Analysis of the restriction pattern of MMTV proviral genomes in mammary tumor cellular DNA showed that four out of 18 virus positive tumors contained common virus-host junction fragments. In each case the MMTV common integration regions (designated Int-1 and Int-2) defined in mammary tumors of inbred mice were unoccupied. We tentatively conclude that the common virus-host junction fragments identified in this study represent a new common integration region for MMTV in mammary tumors.